| Protein misfolding and aggregation are the causes of several neurodegenerative diseases which affect an increasing number of people (i.e., Alzheimer's, Parkinson's, Huntington's and prion disease). In most cases, the mechanism of protein aggregation in solution has been studied in detail, while in cells the mechanism remains unknown, especially because of the difficulty to observe intermediates of aggregation (oligomers) directly in live cells. The techniques used until now to determine the aggregation stages are antibodies labeling and electron microscopy, requiring lysing or fixation of cells.;In this work, a new method to observe aggregation in live cells is introduced: Number and Brightness (N&B) fluctuation spectroscopy analysis. For the first time, this technique has been used to observe, map and quantify the aggregation of proteins in live cells. In particular, the aggregation of the exon1 of Huntingtin (Httex1p) model system of Huntington's disease (HD), has been studied. Huntingtin is the protein responsible for HD, a late-onset neurodegenerative disease. Using N&B fluctuation spectroscopy analysis performed simultaneously on the entire cell, it is possible to observe, for periods of hours, the kinetics of aggregate formation. The N&B analysis method is a very powerful in determining the aggregate size and localization of the aggregates, and in establishing a model for Httex1p aggregation in live cell.;To our knowledge, this is the first time oligomers have been observed, quantified and localized in live cells.;Other fluorescence methods (i.e. FRAP, FCS, FLIM) have been applied to study the structure of the inclusion bodies, the diffusion and the interaction of Httex1p in the cell. We were able to observe the recruitment of Httex1p by the inclusion body from all the compartments of the cell. FLIM highlighted the presence of a "phasor-fingerprint" of the inclusion bodies both in tissues and cells, suggesting that multiple scattering in the inclusion body results in a delay in the emission.;The information obtained from the different fluorescence-based techniques gives a better understanding of Httex1p dynamic in live cells. In this work, we exploit the combination of different fluorescence methods to obtain a comprehensive description of the aggregation process in live cells. |
